U.S. patent number 7,217,419 [Application Number 10/508,222] was granted by the patent office on 2007-05-15 for vaccine composition comprising virus-like particles of human papillomavirus.
This patent grant is currently assigned to GlaxoSmithKline Biologicals SA. Invention is credited to Martine Anne Cecile Wettendorff.
United States Patent |
7,217,419 |
Wettendorff |
May 15, 2007 |
Vaccine composition comprising virus-like particles of human
papillomavirus
Abstract
The present invention relates to a vaccine composition
comprising VLPs containing L1 proteins or functional L1 protein
derivatives from HPV 16, HPV 18, HPV 31 and HPV 45 genotypes.
Inventors: |
Wettendorff; Martine Anne
Cecile (Rixensart, BE) |
Assignee: |
GlaxoSmithKline Biologicals SA
(Rixensart, BE)
|
Family
ID: |
9933201 |
Appl.
No.: |
10/508,222 |
Filed: |
March 17, 2003 |
PCT
Filed: |
March 17, 2003 |
PCT No.: |
PCT/EP03/02826 |
371(c)(1),(2),(4) Date: |
March 31, 2005 |
PCT
Pub. No.: |
WO03/077942 |
PCT
Pub. Date: |
September 25, 2003 |
Prior Publication Data
|
|
|
|
Document
Identifier |
Publication Date |
|
US 20050175632 A1 |
Aug 11, 2005 |
|
Foreign Application Priority Data
|
|
|
|
|
Mar 18, 2002 [GB] |
|
|
0206360.0 |
|
Current U.S.
Class: |
424/204.1;
435/69.1; 424/202.1; 435/5 |
Current CPC
Class: |
A61P
31/12 (20180101); C07K 14/005 (20130101); A61P
15/00 (20180101); A61K 39/12 (20130101); A61P
35/00 (20180101); A61P 31/20 (20180101); A61P
37/04 (20180101); A61P 31/18 (20180101); C12N
7/00 (20130101); A61K 2039/70 (20130101); A61K
2039/55572 (20130101); C12N 2710/20034 (20130101); C12N
2710/20023 (20130101); C12N 2710/20022 (20130101); A61K
2039/55505 (20130101); A61K 2039/5258 (20130101) |
Current International
Class: |
A61K
39/12 (20060101) |
Field of
Search: |
;424/204.1,202.1
;435/69.1,6 ;536/23.72 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
|
|
|
|
|
|
|
WO95/31532 |
|
Nov 1995 |
|
WO |
|
WO 99/13056 |
|
Mar 1999 |
|
WO |
|
WO00/09671 |
|
Feb 2000 |
|
WO |
|
WO01/17551 |
|
Mar 2001 |
|
WO |
|
Other References
Wheeler, "Preventive vaccines for cervical cancer" Salud Publica de
Mexico 39(4) pp. 283-287 (1997). cited by other .
Giuliano et al. "Human papillomavirus infection at the United
States-Mexico Border: Implications for cervical cancer prevention
and control" Cancer Epidemiology Biomarkers & Prevention.
10(11) pp. 1129-1136 (2001). cited by other .
Roden etal. "Assesment of serological relatedness of genital human
papillomaviruses by hemagglutination inhibition" J of Virology, The
American Society for Microbiology 70(5) pp. 1996-2005 (1996). cited
by other .
Palker et al. "Antibody, cytokine and cytotoxic T lymphocyte
responses in chimpanzees immunized with human papillomavirus
virus-like particles" Vaccine, Butterworth Scientific 19(27) pp.
3733-3743 (2001). cited by other.
|
Primary Examiner: Salimi; Ali R.
Attorney, Agent or Firm: Majarian; William R. Venetianer;
Stephen Kinzig; Charles
Claims
The invention claimed is:
1. A vaccine composition comprising an HPV 16 VLP; an HPV 18 VLP;
an HPV 31 VLP; an HPV 45 VLP; and an adjuvant.
2. A vaccine composition comprising an HPV 16 VLP which comprises
an HPV 16 L1 protein or an HPV 16 functional L1 protein derivative,
an HPV 18 VLP which comprises an HPV 18 L1 protein or an HPV 18
functional L1 protein derivative, an HPV 31 VLP comprises an HPV 31
L1 protein or an HPV 31 functional L1 protein derivative, an HPV 45
VLP which comprises an HPV 45 L1 protein or an HPV 45 functional L1
protein derivative, and an adjuvant.
3. The vaccine composition according to claim 1 wherein the HPV 16
VLP consists of an HPV 16 L1 protein or an HPV 16 functional L1
protein derivative, the HPV 18 VLP consists of an HPV 18 L1 protein
or an HPV 18 functional L1 protein derivative, the HPV 31 VLP
consists of an HPV 31 L1 protein or an HPV 31 functional L1 protein
derivative and the HPV 45 VLP consists of an HPV 45 L1 protein or
an HPV 45 functional L1 protein derivative.
4. The vaccine composition according to claim 1 wherein at least
one VLP comprises a truncated L1 protein.
5. The vaccine composition according to claim 1 wherein the
composition further comprises a one or more VLPs from one or more
additional HPV genotvpes.
6. The vaccine composition according to claim 5 wherein the one or
more VLPs are selected from the group consisting of an HPV 33 VLP,
an HPV 35 VLP, an HPV 52 VLP, an HPV 53 VLP, an HPV 56 VLP, an HPV
58 VLP and an HPV 59 VLP.
7. The vaccine composition according to claim 1 which is at least
60% effective in preventing cervical cancer.
8. The vaccine composition according to claim 1 further comprising
an HPV early antigen or immunologically active fragment thereof,
the antigen selected from the group consisting of E1, E2, E3, E4,
E5, E6, E7 and E8.
9. The vaccine composition according to claim 1 formulated with an
antigen derived from an organism causing a sexually transmitted
disease.
10. The vaccine composition according to claim 9 wherein the
antigen is an HSV antigen or immunologically active fragment
thereof.
11. The vaccine composition according to claim 9 wherein the
antigen is a chlamydia antigen or immunologically active fragment
thereof.
12. The vaccine composition according to claim 1 wherein at least
one VLP further comprises an HPV L2 protein or fragment
thereof.
13. The vaccine composition according to claim 1 wherein the
adjuvant comprises an aluminium salt.
14. The vaccine composition according to claim 1 wherein the
adjuvant comprises aluminium hydroxide.
15. The vaccine composition according to claim 1 wherein the
adjuvant comprises 3D-MPL.
16. A method of making the vaccine composition of claim 1, the
method comprising combining an HPV 16 VLP, an HPV 18 VLP, an HPV 31
VLP, an HPV 45 VLP, and an adjuvant.
17. A method of prevention of HPV infection or treatment of HPV,
the method comprising delivering to an individual at risk of
infection an effective amount of a vaccine composition according to
claim 1.
18. The method of claim 16 wherein the VLPs are adsorbed onto
aluminium hydroxide before combining.
19. The vaccine composition according claim 1 wherein the antibody
response against a given VLP type in the vaccine is at least 50%
that of the antibody response of that same VLP type when measured
individually.
20. The vaccine composition according claim 1 wherein the immune
response generated by the vaccine is at a level in which the
protective effect of each VLP type is still seen.
21. The vaccine composition according to claim 1 wherein the
adjuvant comprises an aluminium salt and 3D-MPL.
22. The vaccine composition according to claim 1 wherein the
adjuvant comprises QS21.
Description
The present invention relates to vaccines against HPV. In
particular the invention relates to vaccines comprising virus like
particles (VLPs), especially virus like particles comprising
proteins from human papilloma virus (HPV).
Papillomaviruses are small DNA tumour viruses, which are highly
species specific. So far, over 100 individual human papillomavirus
(HPV) genotypes have been described. HPVs are generally specific
either for the skin (e.g. HPV-1 and -2) or mucosal surfaces (e.g.
HPV-6 and -11) and usually cause benign tumours (warts) that
persist for several months or years. Such benign tumours may be
distressing for the individuals concerned but tend not to be life
threatening, with a few exceptions.
Some HPVs are also associated with cancers. The strongest positive
association between an HPV and human cancer is that which exists
between HPV-16 and HPV-18 and cervical carcinoma. Cervical cancer
is the most common malignancy in developing countries, with about
500,000 new cases occurring in the world each year. It is now
technically feasible to actively combat primary HPV-16 infections,
and even established HPV-16-containing cancers, using vaccines. For
a review on the prospects for prophylactic and therapeutic
vaccination against HPV-16 see Cason J., Clin. Immunother. 1994;
1(4) 293 306 and Hagenesee M. E., Infections in Medicine 1997 14(7)
555 556, 559 564.
Although minor variations do occur, all HPVs genomes described have
at least eight early genes, E1 to E8 and two late genes L1 and L2.
In addition, an upstream regulatory region harbors the regulatory
sequences which appear to control most transcriptional events of
the HPV genome.
HPV L1 based vaccines are disclosed in WO94/00152, WO94/20137,
WO93/02184 and WO94/05792. Such a vaccine can comprise the L1
antigen as a monomer, a capsomer or a virus like particle. Methods
for the preparation of VLPs are well known in the art, and include
VLP disassembly-reassembly approaches to provide enhanced
homogeneity, for example as described in WO9913056 and U.S. Pat.
No. 6,245,568. Such particles may additionally comprise L2
proteins. L2 based vaccines are described, for example, in
WO93/00436. Other HPV vaccines are based on the Early proteins,
such as E7 or fusion proteins such as L2-E7.
Despite the work on HPV vaccines there is still no broadly
effective vaccine against cervical cancer.
The present invention relates to an improved vaccine against human
papilloma virus.
In a first aspect the present invention relates to a vaccine
composition comprising VLPs containing L1 proteins or functional L1
protein derivatives from HPV 16, HPV 18, HPV 31 and HPV 45.
The invention also relates to a method of vaccine production, the
method comprising combining VLPs containing L1 proteins or
functional L1 protein derivatives from HPV 16, HPV 18, HPV 31 and
HPV 45.
The invention further relates to use of a mixture of VLPs
containing L1 proteins or functional L1 protein derivatives from
HPV 16, HPV 18, HPV 31 and HPV 45 in the preparation of a vaccine
for the prevention of cervical cancer.
The invention further relates to a method of preventing cervical
cancer, the method comprising delivering to an individual at risk
of cervical cancer an effective amount of a vaccine as described
above, such as a vaccine comprising a mixture of HPV 16, HPV 18,
HPV31 and HPV 45 VLPs.
The VLPs of the present invention can be formed from either the
full length HPV L1 protein or certain L1 derivatives using standard
methods in the art, for example as disclosed in WO99/13056
incorporated herein by reference.
It is preferred that the L1 protein used to form the VLP is a
truncated L1 protein. Preferably at least one of the VLPs comprises
a truncated L1 protein, and preferably all the L1 proteins in the
combination vaccine are truncated L1 proteins. Preferably the
truncation removes a nuclear localisation signal. Preferably the
truncation is a C-terminal truncation. Preferably the C-terminal
truncation removes fewer than 50 amino acids, more preferably fewer
than 40 amino acids. Most preferably the C terminal truncation
removes 34 amino acids from HPV 16 and 35 amino acids from HPV
18.
Truncated L1 proteins are suitably functional L1 protein
derivatives. Functional L1 protein derivatives are capable of
raising an immune response (if necessary, when suitably
adjuvanted), said immune response being capable of recognising a
VLP consisting of the full length L1 protein and/or the HPV type
from which the L1 protein was derived.
VLPs of the invention may also comprise other types of functional
protein derivatives, including mutants of the full length or
truncated HPV L1 proteins such as deletion, substitution, or
insertion mutants. Suitable derivatives also include codon
optimised sequences. The L1 protein or derivative may also be a
fusion protein, such as the fusion of the L1 protein with L2 or an
early protein. Preferably fusion proteins comprise proteins from
only one HPV genotype. VLPs made from chimaeric L1 proteins in
which L1 proteins from one genotype are linked to L1 proteins from
other genotypes are not preferred.
The L1 protein or functional protein derivative is suitably able to
form a VLP, and VLP formation can be assessed by standard
techniques such as, for example, electron microscopy and dynamic
laser light scattering.
Preferably the polydispersity of the VLPs is less than 0.15, most
preferably less than 0.1 and more preferably less than 0.08 when
measured using a Malvem Zetasizer 3000HS under conditions as
described herein.
Use of the term `protein` or reference to a specific protein e.g.
`L1` is hereinafter taken to include reference to functional
protein derivatives, unless otherwise indicated or obviously
apparent from the context.
In a preferred aspect of the invention the vaccine of the invention
has only four VLP types--HPV 16, HPV 18, HPV 31 and HPV 45 VLPs.
Preferably the VLPs are L1--only VLPs from each of these 4
genotypes.
Alternatively, and most preferred, the vaccine comprises an
additional HPV valency, making a pentavalent vaccine. Preferably
the additional valency is a VLP comprising an L1 protein or
functional derivative, as above, from one of HPV 52, 53, 58, 33,
35, 56, and 59. Preferably the 5.sup.th genotype is HPV 33 when the
vaccine is for use in South America or HPV 52, 53 or 58 when the
vaccine is for use in Asia.
The present invention also extends to vaccines comprising 2 or more
additional valencies, to provide a vaccine with 6 or more
genotypes.
In one preferred embodiment the combination excludes VLPs from HPV
6a, 6b or HPV 11 genotypes.
Preferably the vaccine of the invention is at least 55% effective
in preventing cervical cancer, more preferably 60%, 65%, 70%, 75%
preferably 80% or even more effective in prevention of cervical
cancer. For the avoidance of doubt, % efficacy in prevention of
cervical cancer means protection against all cervical cancer
induced by HPV infection, and not just protection against cancer
caused by one genotype. Prevention may be suitably assessed over 1
year post initial vaccination, although preferred vaccines are
equally effective over 2, 3, 4, 5 or more years. The % efficacy can
be increased by selecting appropriate HPV genotypes to target the
vaccine formulation to specific geographical areas.
Preferably the combination of VLPs within the vaccine does not
reduce the immunogenicity of each VLP type. In particular it is
preferred that there is no interference between HPV VLPs in the
combination of the invention, such that the combined VLP vaccine of
the invention is able to offer effective protection against
infection by each HPV genotype represented in the vaccine. Suitably
the immune response against a given VLP type in the combination is
at least 50% of the immune response of that same VLP type when
measured individually, preferably 100% or substantially 100%. For
responses to the HPV 16 and HPV 18 VLPs, the combined vaccine of
the invention preferably stimulates an immune response which is at
least 50% of that provided by a combined HPV 16/HPV 18 VLP vaccine.
Suitably the immune response generated by the vaccine of the
invention is at a level in which the protective effect of each VLP
type is still seen. The immune response may suitably be measured,
for example, by antibody responses, as illustrated herein.
The vaccine of the invention may be used to treat or prevent HPV
infection and/or disease. For example the vaccine may be used
therapeutically to reduce viral load and/or infections that lead to
cervical carcinoma or CIN III sequelae. The invention thus relates
to use of the vaccine of the invention in the therapeutic treatment
of diseases related to HPV infection and in prophylaxis of
infection or disease. The invention also relates to use of the VLP
combination of the invention in generation of an immune response
against HPV 16, 18, 31 and 45.
The vaccine of the invention may optionally be formulated with VLPs
which provide protection against genital warts, such as VLPs
containing L1 protein from HPV 6a, 6b and/or HPV 11 genotypes.
Preferably the VLPs comprise the HPV L1 protein only and no L2
protein or protein fragment.
Vaccines of the invention may comprise other proteins or protein
fragments in addition to the L1 protein or derivative.
Proteins/peptides may be delivered in chimaeric form with the L1
protein in the VLP, encapsulated within a VLP or co-formulated in a
mixture with the VLP's. Other proteins or peptides may also be
co-administered with the vaccine of the invention.
In one aspect the vaccine comprises an HPV L2 protein or L2
derivative such as an L2 peptide, for example as disclosed in K.
Kawana et al Vaccine 19, (2001) p1496 1502, incorporated herein by
reference. In a further preferred embodiment the vaccine of the
invention may be formulated with HPV early antigens such as E1, E2,
E3, E4, E5, E6, E7, E8 or immunologically active derivatives
thereof. When delivered in chimaeric form it is preferable to
utilise an immunogenic fragment of about 30 60 amino acids of the
early antigen.
Optionally the vaccine may also be formulated or co-administered
with non-HPV antigens. Suitably these antigens can provide
protection against other diseases, most preferably sexually
transmitted diseases such as herpes simplex virus, chlamydia and
HIV. We particularly prefer that the vaccine comprises gD or a
truncate thereof from HSV, preferably the gD2t protein as described
in WO 99/45957. In this way the vaccine provides protection against
both HPV and HSV. Preferred HIV antigens are described in
WO/9916884 and WO/0154719.
The present invention generally relates to a mixture of VLPs
containing capsid proteins from HPV16, 18, 31 and 45, such as
L1-only VLPs.
In a particularly preferred embodiment, the invention provides a
vaccine comprising a mixture of HPV 16 VLPs, HPV 18 VLPs, HPV 31
VLPs and HPV 45 VLPs. Reference herein to HPV 16 VLPs, for example,
is a reference to an L1 VLP wherein the L1 protein or L1 derivative
is from HPV 16. The same nomenclature principle applies, by
extension, to other VLPs described herein, such as HPV 18, HPV 31
and HPV 45 VLPs.
Preferably each VLP contains L1 protein from only 1 HPV genotype.
Such a vaccine may be formulated by production of individual VLPs
from HPV 16, 18, 31 and 45, followed by combination of such VLPs.
Preferably there are no other HPV proteins in the VLP other than
L1.
Also preferred are VLPs containing proteins from only one HPV
genotype, such as VLPs with L1 and L2 from HPV 16.
However, in an alternative embodiment of the invention, the VLPs
may be mixed VLPs, a mixed VLP comprising L1 protein from one
genotype in combination with L1 protein from a second genotype,
wherein the different L1 proteins are not chimaeric L1 proteins,
but associate together within the same capsid structure to form
immunogenic VLPs.
Preferred combinations include any permutation of genotypes 16, 18,
31 and 45--for example, the invention may comprise a mixed HPV
16/HPV 18 VLP in combination with a mixed HPV31/HPV 45 VLP, or
mixed 16/31 VLPS in combination with mixed 18/45 VLPs. Combinations
of more than 2 L1 genotypes within 1 VLP are also contemplated.
Mixed VLPs may be produced by separate expression of the individual
L1 proteins followed by combination to form VLPs, as exemplified
herein. Alternatively multiple L1 proteins may be expressed within
the same cell, from one or more DNA constructs. For example,
multiple DNA constructs may be transformed or transfected into host
cells, each vector encoding a different L1 protein. Alternatively a
single vector having multiple L1 genes, controlled by a shared
promoter or multiple individual promoters, may be used. IRES
elements may also be incorporated into the vector, where
appropriate. Using such expression strategies the co-expressed L1
proteins may be co-purified for subsequent VLP formation, or may
spontaneously form mixed VLPs which can then be purified.
Where mixed VLPs are used, a preferred process for mixed VLP
production comprises preparation of VLP L1 proteins or derivatives,
such as L1 proteins, from different papillomavirus genotypes,
mixing the proteins if necessary and assembly of the proteins to
produce mixed VLPs. The L1 proteins may be in the form of a crude
extract, be partially purified or purified prior to mixing.
Preferably the proteins are at least partially purified before
being combined. Optionally, further purification of the mixed VLPs
may be carried out after assembly. Where additional antigens are
used, then these may be added where appropriate.
In one embodiment mixed VLPs may be made by disassembly of 2 or
more VLPs, followed by combination of the disassembled VLP
components at any suitable point prior to reassembly. This approach
is suitable when VLPs spontaneously form when the L1 protein is
expressed, as occurs for example, in some yeast strains. Where the
expression of the L1 protein does not lead to spontaneous VLP
formation, preparations of L1 proteins or capsomers may be combined
before assembly into VLPs.
Assembly of VLPs is generally achieved by removal of a reducing
agent. As such, in mixed VLP production, the mixing of proteins
preferably takes place prior to the removal of a reducing agent
from the mixture of proteins. Preferably the production of mixed
VLPs comprises the step of mixed VLP formation from a mixture of
dissociated L1 proteins by removal of a reducing agent from the
mixture under conditions that allow VLPs to form.
Preferably the reassembly process results from removal of a
reducing agent such as .beta.-mercaptoethanol.
It is, however, known that VLP formation is dependent upon pH,
metal ions and salinity as well as the presence of a reducing
agent. As such, under certain circumstances, it may be envisaged
that VLPs might form in the presence of a reducing agent. It is
only important to the invention that mixing of the proteins from
different genotypes occurs prior to the change in environmental
condition that allows the mixed VLPs to form, whether this is pH,
metal ions, salinity, reducing environment or combination of
these.
Where mixed VLPs are used, preferably the components of the VLPs
are mixed in the proportions in which they are desired in the final
mixed VLP. For example, a mixture of the same amount of a partially
purified L1 protein from HPV 16 and HPV 18 provides a mixed VLP
with approximately equal amounts of each protein.
Vaccine solutions comprising mixed VLPs may be stabilised by
compositions known in the art, such as those of WO 98/44944,
WO0045841, incorporated herein by reference.
For all vaccines of the invention, it is preferred that the vaccine
is used for the vaccination of adolescent girls aged 10 15,
preferably 10 13 years. The vaccine may also be administered to
women following an abnormal pap smear or after surgery following
removal of a lesion caused by HPV.
Preferably the vaccine is delivered in a 2 or 3 dose regime, for
example in a 0, 1 month regime or 0, 1 and 6 month regime
respectively. Suitably the vaccination regime incorporates a
booster injection after 5 to 10 years, preferably 10 years.
Preferably the vaccine is a liquid vaccine formulation, although
the vaccine may be lyophilised and reconstituted prior to
administration.
The vaccines of the invention may also comprise adjuvants in
combination with the VLPs. Suitably the VLPs of the invention are
used in combination with aluminium, and are suitably adsorbed or
partially adsorbed onto aluminium adjuvants. Also preferred are
adjuvants which stimulate a Th1 type response such as 3DMPL or
QS21. Suitably the adjuvant is an aluminium salt, preferably in
combination with 3D MPL, such as aluminium phosphate and
3D-MPL.
A preferred adjuvant is aluminium hydroxide, with the combination
of aluminium hydroxide with 3D-MPL especially preferred.
When VLPs are adsorbed on to aluminium containing adjuvants, the
adjuvant is preferably added before mixing of the VLPs to form the
final vaccine product.
The vaccine may also comprise aluminium or an aluminium compound as
a stabiliser, and the present invention also relates to a
stabilised combination vaccine wherein the VLPs are adsorbed onto
an aluminium salt. Suitably the VLPs are more stable over time
after adsorption onto an aluminium salt than in the absence of
aluminium. Preferably stabilised VLPs are obtained or obtainable by
methods according to example 1 section C3.
The vaccines of the invention may be provided by any of a variety
of routes such as oral, topical, subcutaneous, musosal (typically
intravaginal), intraveneous, intramuscular, intranasal, sublingual,
intradermal and via suppository. Intramuscular and intradermal
delivery are preferred.
The dosage of VLP and other proteins will vary with the condition,
sex, age and weight of the individual, the administration route and
HPV of the vaccine. The quantity may also be varied with the number
of VLP types. Suitably the delivery is of an amount of VLP suitable
to generate an immunologically protective response. Suitably each
vaccine dose comprises 1 100 .mu.g of each VLP, preferably 5 80
.mu.g, more preferably 5 30 .mu.g each VLP, most preferably 5 20
.mu.g of each VLP with 5 .mu.g, 6 .mu.g, 10 .mu.g, 15 .mu.g or 20
.mu.g especially preferred.
The multivalent vaccine of the present invention is suitably
produced by combining purified L1 VLPs. Methods for the production
of L1 VLPs are well known in the art, and include for example
methods given in WO9531532, WO9615247, WO00/09671 and U.S. Pat. No.
5,888,526, the whole contents of which are incorporated herein.
Suitably the VLPs of the invention are made by disassembly and
reassembly of VLPs, to provide homogenous and pure VLPs. Examples
of suitable processes are given in WO0057906, U.S. Pat. No.
6,245,568 and WO9913056.
Preferably the VLPs are prepared from insect cells such as Sf9 or
Hi-5 cells, although any suitable cells such as E. coli or yeast
cells, for example, S. cerevisiae S. pombe or Pichia sp. may also
be used.
Preferably the purification of VLPs after L1 expression includes
one or more of the steps of anion exchange chromatography (Di
methyl amino ethyl--DMAE), anion exchange chromatography (tri
methyl amino ethyl--TMAE), hydroxyapatite chromatography,
filtration such as nanometric filtration or ultrafiltration, or
hydrophobic interaction chromatography. Preferably at least one
anion exchange step is performed during purification, and more
preferably 2 anion exchange steps are used. Preferably at least one
anion exchange purification step is performed prior to mixing the
proteins. Optionally a UV irradiation step may be employed.
For the avoidance of doubt, the entire teaching of all documents
referred to herein is incorporated by reference.
The present invention is illustrated by the following non-limiting
Examples and Figures, wherein:
FIG. 1 illustrates mixed VLPs in comparison with HPV 16 VLPs as
assessed by EM;
FIGS. 2 and 3 illustrate size distribution of mixed VLPs;
FIG. 4 illustrates antibody responses against VLP 16 in a mixed HPV
16, 18, 31, 45 combination vaccine vs. an HPV 16 control;
FIG. 5 illustrates antibody responses against VLP 18 in a mixed HPV
16, 18, 31, 45 combination vaccine vs. an HPV 18 control;
FIG. 6 illustrates antibody responses against VLP 31 in a mixed HPV
16, 18, 31, 45 combination vaccine vs. an HPV 31 control; and
FIG. 7 illustrates antibody responses against VLP 45 in a mixed HPV
16, 18, 31, 45 combination vaccine vs. an HPV 45 control.
EXAMPLE 1
The combination of HPV 16 and HPV 18 L1 VLPs is detailed herein. L1
proteins from other HPV genotypes may be readily produced by
similar methods, already known in the art.
A Preparation of HPV 16/18 L1 VLPs
Production of HPV 16 and HPV 18 VLPs was carried out using standard
protocols--for example, see WO9913056. HPV 16/18 proteins were
expressed in Trichoplusia ni (High Five.TM.) cells (at a density of
.about.350000 cells/ml) infected with recombinant Baculovirus (MOI
of 0.3) encoding the HPV 16 or 18 .mu.l gene of interest. Cells
were harvested approximately 72 hours post infection.
B Cell Harvest/Antigen Extraction
The antigen (L1-16/18) was extracted from Hi5 cells in a three step
process of concentration, extraction, clarification. The
concentration step consists removes up to 90% of the culture
medium, and was performed by tangential flow filtration. The
extraction step was performed with a hypotonic buffer (Tris 20 mM,
pH 8.5). A volume equal to the culture volume was used to perform
the extraction. A contact time of minimum half an hour under smooth
agitation was used. The clarification was performed by tangential
flow filtration.
C Purification
The purification process was carried out at room temperature.
.beta.-mercaptoethanol (4% w/w) was added to the extract in order
to disassemble the VLP's into capsomers, for both antigens,
L1-16/18. Glycerol was added up to a concentration of w/w 10% just
prior to the addition of .beta.-mercaptoethanol.
All buffers used were filtered on 0.22 .mu.m filters prior to
storage at 2.degree. C. 8.degree. C. Prior to each purification
run, gel matrixes are sanitised and equilibrated with appropriate
buffer before sample loading.
Purification regiemes are given for the separate purification of L1
from both HPV 16 and 18. These schemes are broadly similar, and
involve the steps of: Anion exchange chromatography (Di methyl
amino ethyl--DMAE), Anion exchange chromatography (tri methyl amino
ethyl--TMAE), Hydroxyapatite chromatography, Nanometric filtration
(Planova), Ultrafiltration, Hydrophobic interaction chromatography
(using Octyl Sepharose) for HPV 18 or Anion exchange chromatography
(DEAE) for HPV 16; and Sterile filtration. Specifically: C1
Purification of L1-18 Antigen Anion Exchange Chromatography
DMAE
The clarified extract (protein at a concentration of .about.1 g/ml,
with the L1 protein at .about.150 mg/ml) is applied to an anion
exchange column (Di Methyl Amino Ethyl). Elution is performed with
(Tris 20 mM|NaCl 200 mM|4% .beta.-mercaptoethanol BME) buffer, pH
7.9.+-.0.2. The antigen is eluted in approximately 5 column volumes
and the elution profile is monitored at 280 nm.
Anion Exchange Chromatography TMAE
The eluate of the first step is diluted with 1 volume of
H.sub.2O/BME 4%. The diluted eluate is then applied to a second
anion exchange column (Tri Methyl Amino Ethyl). Elution is
performed with (20 mM Tris|NaCl 200 mM|4% BME) buffer, pH
7.9.+-.0.2. The antigen is eluted in approximately 4 column volumes
and the elution profile is monitored at 280 nm.
Hydroxyapatite Chromatography
The eluate of the TMAE step is applied to a hydroxyapatite (HA)
column. After sample application, the gel is eluted with
approximately 2.5 column volumes of (NaH.sub.2PO.sub.4 100 mM|NaCl
30 mM|4% BME) buffer, pH 6.0.+-.0.2.
Nanometric Filtration (Planova)
The HA eluate is diluted in order to reach the following
conditions: (NaH.sub.2PO.sub.4 25 mM|NaCl 10 mM|4% BME) buffer, pH
7.5.+-.0.2.
Then it is filtered successively on a 0.2 .mu.m prefilter and on a
Planova 15N filter of 0.12 m.sup.2. The filtration is performed at
constant pressure 200 mbar.+-.20 mbar.
Ultrafiltration
The ultrafiltration is performed with a tangential flow
ultrafiltration system equipped with polyethersulfone membranes
(Centramate cassette 0.1 m.sup.2, 100 kD).
The Planova eluate is treated to reach the following conditions:
(NaH.sub.2PO.sub.4 100 mM|NaCl 30 mM|4% BME), pH 6.0.+-.0.2; then
it is loaded in the system, concentrated 5 fold and dia-filtrated
with continuous injection of .about.10 starting volumes of
(NaH.sub.2PO.sub.4 20 mM|NaCl 500 mM) buffer, pH 6.0.+-.0.2.
Hydrophobic Interaction Chromatography (Octyl Sepharose)
The ultrafiltration permeate is applied to an Octyl Sepharose
column.
This chromatography step is run in the negative mode with
approximately 5 column volumes of (Na.sub.3PO.sub.4 20 mM|NaCl 500
mM) buffer, pH 6.0.+-.0.2.
Sterile Filtration
The purified L1-18 antigen solution is sterilised by filtration on
a 0.22 .mu.m membrane.
C2 Purification of L1-16 Antigen
Anion Exchange Chromatography DMAE
The clarified extract is applied to an anion exchange column (Di
Methyl Amino Ethyl).
Elution is performed with (Tris 20 mM|NaCl 180 mM|4% BME) buffer,
pH 7.9.+-.0.2. The antigen is eluted in approximately 4 column
volumes and the elution profile is monitored at 280 nm.
Anion Exchange Chromatography TMAE
The eluate of the first step is diluted with 1 volume of
H.sub.2O/BME 4%. The diluted eluate is then applied to a second
anion exchange column (Tri Methyl Amino Ethyl). Elution is
performed with (20 mM Tris|NaCl 180 mM|4% BME) buffer, pH
7.9.+-.0.2. The antigen is eluted in approximately 5 column volumes
and the elution profile is monitored at 280 nm.
Hydroxyapatite Chromatography (HA)
The eluate of the TMAE step is applied to a HA column.
After sample application, the gel is eluted with approximately 3
column volumes of (NaH.sub.2PO.sub.4 100 mM|NaCl 30 mM|4% BME)
buffer, pH 6.0.+-.0.2.
Nanometric Filtration (Planova)
The HA eluate is diluted in order to reach the following
conditions: (NaH.sub.2PO.sub.4 25 mM|NaCl 10 mM|4% BME) buffer, pH
7.5.+-.0.2.
Then it is filtered successively on a 0.2 .mu.m prefilter and on a
Planova 15N filter of 0.12 m.sup.2. The filtration is performed at
constant pressure 200 mbar.+-.20 mbar.
Ultrafiltration
The ultrafiltration is performed with a tangential flow
ultrafiltration system equipped with polyethersulfone membranes
(Centramate cassette 0.1 m.sup.2, 100 kD).
The Planova eluate is treated to reach the following conditions:
(NaH.sub.2PO.sub.4 100 mM|NaCl 30 mM|4% BME), pH 6.0.+-.0.2; then
it is loaded in the system, concentrated 5 fold and dia-filtrated
with continuous injection of .about.10 starting volumes of
(NaH.sub.2PO.sub.4 20 mM|NaCl 500 mM) buffer, pH 6.0.+-.0.2.
Anion Exchange Chromatography DEAE
The ultrafiltration eluate is adjusted to the conductivity of the
equilibrium buffer, (Na.sub.3PO.sub.4 20 mM|NaCl 250 mM), pH
6.0.+-.0.2 and applied on an anion exchange column (Di Ethyl Amino
Ethyl).
Elution is performed with (NaH.sub.2PO.sub.4 20 mM|NaCl 500 mM)
buffer, pH 6.0.+-.0.2. The antigen is eluted in approximately 3
column volumes and the elution profile is monitored at 280 nm.
Sterile Filtration
The purified L1-16 antigen solution is sterilised by filtration on
a 0.22 .mu.m membrane.
C3
Each VLP type is adsorbed independently to produce a concentrated
adsorbed monovalent.
Preparation of VLP16 concentrated adsorbed monovalent:
60 .mu.g of purified VLPs from HPV16 are adsorbed on 150 .mu.g
Al.sup.3+ from Al(OH).sub.3, at a pH of 6.0.+-.0.2, for one hour at
room temperature with gentle stirring. This concentrated adsorbed
monovalent is stored at +4.degree. C. Adsorption is checked by
centrifuging the preparation and quantifying VLPs in the
supernatant.
Preparation of VLP18 concentrated adsorbed monovalent:
60 .mu.g of purified VLPs from HPV18 are adsorbed on 150 .mu.g
Al.sup.3+ from Al(OH).sub.3, at a pH of 6.0.+-.0.2, for one hour at
room temperature with gentle stirring. This concentrated adsorbed
monovalent is stored at +4.degree. C. Adsorption is checked by
centrifuging the preparation and quantifying VLPs in the
supernatant.
D Final Vaccine Preparation:
Concentrated adsorbed monovalents prepared by the above method were
combined to form a suspension containing 20 .mu.g each VLP per
dose. Final vaccine is stored at +4.degree. C.
Addition of VLPs from HPV 31 and 45 at a concentration of 20 .mu.g
each VLP completes the tetravalent vaccine.
The combined adsorbed bulks, or individual adsorbed bulks, may be
further mixed with adjuvants such as 3D-MPL.
EXAMPLE 2
A Preparation of HPV 16/18 L1 VLPs
Production of HPV 16 and HPV 18 VLPs was carried out using standard
protocols--as above.
B Formation of Mixed VLPs
The process of the invention involves dissassembly and then
reassembly of the HPV 16 and 18 VLPs such that the reassembly of
HPV L1 16 and 18 is carried out together to permit the formation of
a mixed VLP.
The HPV 16 and 18 VLPs may be combined at any suitable point in the
above process prior to the point at which the VLPs are
reassembled.
By way of example 2 specific strategies have been tested: 1 mixing
of both antigens after the HA step. Based on the L1 concentration
in HA pools, the two components are mixed to reach an equal
concentration of HPV16 and 18 to start the UF step. In this case
after the ultrafiltration step an Octyl speharose step is performed
as for HPV 18 purification followed by a DEAE step as performed in
the HPV 16 procedure. 2 mixing of both extracts and copurify. Based
on the L1 concentration in Extracts, the two valences are mixed to
reach an equal concentration of HPV16 and 18 to start the DMAE
step. Again, after the ultrafiltration step an Octyl speharose step
is performed as for HPV 18 followed by a DEAE step as performed in
the HPV 16 procedure.
##STR00001## HPV16-HPV18 VLP-Mixed at DMAE Step
The same flow sheet is applied but the mixing is performed at the
DMAE step instead of the UF step. The concentration used for
elution at the anion exchange DMAE TMAE steps is 200 mM.
Results
HPV16HPV18 VLP-Mixed at UF Step
2 lots of mixed VLPs (lot numbers 31b165c and 31b166c) were
produced by combining HPV 16 and HPV 18 L1 proteins.
Purity By SDS-Page
The purity of the mixed VLP's was as good as both "classical" HPV
16 or HPV 18 bulks. The purity of the bulks was higher than
95%.
EM Data--FIG. 1
The EM of 31B165C (UF Retentate after maturation) was compared to a
classical HPV16 lot (39B122c). VLP's were well formed, homogeneous
in size, without aggregation; some ribbons of capsomeres are
present in both experiments.
Size Distribution
The size distribution of the VLP's were determined using a Malvern
Zetasizer 3000 HS.
The samples were measured undiluted into a plastic cuvette for
Malvern analysis (800 .mu.l/cuvette).
The technical conditions were: laser wavelength: 532 nm, laser
power: 50 mW, scattered light detected at 90.degree., temperature:
25.degree. C., duration: automatic determination by the software,
number: 3 consecutive measurements, z-average diameter: by
cumulants analysis, size distribution: by the Contin method.
Classical results for HPV18 L1-VLP's are: 70 80 nm with good
polydispersity (<0.1) Classical results for HPV16 L1-VLP's are:
60 70 nm with good polydispersity (<0.1)
For mixed VLP's, the following results were obtained: 31 B165c: 85
nm with good polydispersity (0.08). VLP's are almost completely
formed at the beginning of maturation 31 B166c: 76 nm with good
polydispersity (0.08).
The size distribution of lot 31 B165c and 31 B166c as measured by
dynamic laser light scattering is shown in FIGS. 2 and 3.
HPV16-HPV18 VLP Mixed at DMAE Step
Lot n.degree..31B167B was made up from lots E18L1C005 (HPV18) and
39B167 (HPV16).
Purity By SDS-Page
The purity of the mixed VLP's was as good as both "classical"
bulks.
The purity of the bulks was higher than 95%.
Size Distribution
The size distribution of the VLP's were determined by using a
Malvern Zetasizer 3000 HS. Classical results for HPV18 L1-VLP's
are: 70 80 nm with good polydispersity (<0.1) Classical results
for HPV16 L1-VLP's are: 60 70 nm with good polydispersity
(<0.1)
For mixed VLP's, the following results were obtained: HPV16-HPV18
31B167B: 74 nm with good polydispersity (0.07). VLP's were almost
completely formed at the beginning of maturation
EXAMPLE 3
Production of a Mixed HPV 16, 18, 31, 45 Combination Vaccine
Introduction
An immunogenicity study was performed in Balb/C mice using a
combination of C-terminally truncated L1 VLPs 16, 18, 31 & 45
adjuvanted with alum+3D-MPL (herein `adjuvant A`--50 .mu.g
aluminium salt and 5 .mu.g 3D MPL).
4 groups of 10 mice were immunised twice intramuscularly on day 0
and 21 respectively with: 1. VLP 31 (2 .mu.g)/adjuvant A 2. VLP 45
(2 .mu.g)/adjuvant A 3. VLP 16 (2 .mu.g) and VLP 18 (2
.mu.g)/adjuvant A 4. VLP 16 (2 .mu.g), VLP 18 (2 .mu.g), VLP 31 (2
.mu.g), VLP 45 (2 .mu.g)/adjuvant A
Antibody responses against VLPs 16, 18, 31 and 45 were monitored on
sera taken at day 35 (14 days post dose II).
Results are shown in FIGS. 4 7.
Antibody Response Against VLP 16
Strong antibody responses are induced in post II sera by either
VLP16 formulated with VLP 18 on adjuvant A (group 3) or by the full
combo (group 4) Similar level of antibodies directed against VLP 16
are measured in both groups and no interference is observed.
Antibody Response Against VLP 18 Strong antibody responses are
induced in post II sera by either VLP18 formulated with VLP 16 on
adjuvant A (group 3) or by the full combo (group 4). Similar level
of antibodies directed against VLP 18 are measured in both groups
(less than 1.5 fold difference) and no interference was observed.
Antibody Response Against VLP 31 Strong antibody responses are
induced in post II sera by either VLP31 formulated alone on
adjuvant A (group 1) or by the full combo (group 4). Similar level
of antibodies directed against VLP 31 are measured in both groups,
therefore no interference is observed. Antibody response against
VLP 45 Strong antibody responses are induced in post II by either
VLP 45 formulated alone on adjuvant A (group 2) or by the full
combo (group 4). Similar level of antibodies directed against VLP
45 are measured in both groups, thus no interference is
observed.
CONCLUSIONS
No interference is observed when the four VLPs (VLPs16, 18, 31
& 45) are delivered as a combination.
* * * * *